Project summary Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy with a five-year survival of less than 5%. Genomic analyses of human samples suggest that PDAC can fundamentally be classified into at least two major subtypes based on cancer cell autonomous properties: Classical and Basal-like. These two subtypes have discrete cellular identities, as reflected by the differential expression of several lineage specifiers, i.e. transcription factors that regulate differentiation state. The Classical subtype expresses high levels of lineage specifiers pivotal for foregut endodermal determination (including HNF4A). The Basal-like subtype expresses high levels of transcription factors promoting alternative identities, such as the mesodermal lineage specifiers SIX4 and SIX1. Moreover, the Basal-like subtype confers poor prognosis and responds less well to first-line chemotherapy compared to the Classical subtype. These correlations raise the possibility that these lineage specifiers are not merely markers of PDAC subtype, but might regulate the malignant potential of this disease. Our long-term goal is to identify the molecular regulators of PDAC subtype and thereby identify subtype-specific vulnerabilities that might be exploited therapeutically. A major rationale for this proposal is that the field must achieve a mechanistic understanding of PDAC molecular subtype specification in order to develop such therapeutic strategies. In this proposal, our immediate goal is to test the central hypothesis that a network of lineage specifiers, including HNF4? and SIX1/4, regulates PDAC molecular subtype and malignant potential. We will test this hypothesis in the following specific aims using an integrative approach that employs genetically engineered mouse models, novel organoid culture systems, and patient derived xenografts: (1) Determine the role of distinct HNF4? isoforms in PDAC growth, molecular subtype and drug response. (2) Elucidate the function of SIX1 and SIX4 in the Basal-like subtype of PDAC.